The N17 domain mitigates nuclear toxicity in a novel zebrafish Huntington’s disease model

Abstract

BackgroundAlthough the genetic cause for Huntington’s disease (HD) has been known for over 20 years, the mechanisms that cause the neurotoxicity and behavioral symptoms of this disease are not well understood. One hypothesis is that N-terminal fragments of the HTT protein are the causative agents in HD and that peptide sequences adjacent to the poly-glutamine (Q) repeats modify its toxicity. Here we test the function of the N-terminal 17 amino acids (N17) in the context of the exon 1 fragment of HTT in a novel, inducible zebrafish model of HD.ResultsDeletion of N17 coupled with 97Q expansion (mHTT-ΔN17-exon1) resulted in a robust, rapidly progressing movement deficit, while fish with intact N17 and 97Q expansion (mHTT-exon1) have more delayed-onset movement deficits with slower progression. The level of mHTT-ΔN17-exon1 protein was significantly higher than mHTT-exon1, although the mRNA level of each transgene was marginally different, suggesting that N17 may regulate HTT protein stability in vivo. In addition, cell lineage specific induction of the mHTT-ΔN17-exon1 transgene in neurons was sufficient to recapitulate the consequences of ubiquitous transgene expression. Within neurons, accelerated nuclear accumulation of the toxic HTT fragment was observed in mHTT-ΔN17-exon1 fish, demonstrating that N17 also plays an important role in sub-cellular localization in vivo.ConclusionsWe have developed a novel, inducible zebrafish model of HD. These animals exhibit a progressive movement deficit reminiscent of that seen in other animal models and human patients. Deletion of the N17 terminal amino acids of the huntingtin fragment results in an accelerated HD-like phenotype that may be due to enhanced protein stability and nuclear accumulation of HTT. These transgenic lines will provide a valuable new tool to study mechanisms of HD at the behavioral, cellular, and molecular levels. Future experiments will be focused on identifying genetic modifiers, mechanisms and therapeutics that alleviate polyQ aggregation in the nucleus of neurons.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0063-2) contains supplementary material, which is available to authorized users.

Profilin reduces aggregation and phase separation of huntingtin N-terminal fragments by preferentially binding to soluble monomers and oligomers

“…Recent in vivo studies from Yang and coworkers showed that a ∆N17 variant of huntingtin, which lacks the N17 module, leads to faithful reproduction of HD phenotypes in transgenic mice (56,57). In light of these observations, and given the relative ease of synthesis and purification of Htt-NTFs that lack the N17 module (19), we used constructs of the form Q n -C38 for a majority of our in vitro experiments.…”

Does N‐terminal huntingtin function as a ‘holdase’ for inhibiting cellular protein aggregation?

“…Addition of free N17 was found to accelerate aggregation of 51Q although later reports failed to reproduce these results . Interestingly, the deletion of N17 domain was found to accelerate aggregation of the polyQ domain in transgenic animal models . One possible explanation is the loss of NES (nuclear export signal) sequence which is localized in this domain, leading to the formation of more toxic intranuclear aggregates and explaining the contradictory results observed in vivo .…”

“…Addition of free N17 was found to accelerate aggregation of 51Q [26] although later reports failed to reproduce these results [47,60]. Interestingly, the deletion of N17 domain was found to accelerate aggregation of the polyQ domain in transgenic animal models [48,49]. One possible Table 2.…”

“…The flanking sequences of the polyglutamine tract, including the 17 amino acids at the N terminus of the protein, are known to play a crucial role in the ability of mutant huntingtin (m-htt) to form aggregates [26,[44][45][46][47][48][49]. When full-length huntingtin was expressed in heterologous expression systems, the initiator methionine was missing in all N-terminal peptides and the penultimate alanine was mostly acetylated [50].…”

Huntingtin confers fitness but is not embryonically essential in zebrafish development

“…Zebrafish has been used to model several neurodegenerative disorders due to its genetic conservation to mammals and ease of experimental manipulation [8]. In the context of HD, 1/11 transgenic zebrafish expressing mutant HTT exon 1 fragment exhibit adult-onset movement deficit, which is worsened by the deletion of the N-terminal first 17 amino acids (N17) of mutant HTT fragment [9]. To understand the functions of HTT in development, several studies in zebrafish have utilized antisense morpholinos to knock down HTT transcripts.…”